Our studies provide evidence that a temporal mechanism of γ-globin gene silencing is operative at the −566 Aγ-globin GATA motif. GATA-1 is recruited first, at day E16, followed by the recruitment of FOG-1 and Mi2 at day E17, indicating that assembly of the GATA-1-FOG-1-Mi2 repressor complex occurs sequentially over a 24 hour period. The binding of the GATA-1 repressor complex might change the “transcription-ready” state to a more permanently silenced state by altering the chromatin into a heterochromatic state, preventing γ-globin gene transcription (temporal repression model; ).
Our data also demonstrate that the −566 GATA motif is occupied by GATA-2 early in fetal definitive erythropoiesis (day E12), followed by a change to GATA-1 occupancy at day E16, suggesting that GATA factor occupancy switching may play a role in the silencing of γ-globin expression. GATA-2 is crucial for the maintenance and proliferation of immature hematopoietic progenitors, whereas GATA-1 is essential for the survival of erythroid progenitors and for the terminal differentiation of erythroid cells 
. Changes in global gene expression patterns during hemoglobin switching are accompanied by changes in the expression of GATA-2 and GATA-1 (GATA switching), which in part coordinates cellular maturation 
. These changes in GATA factor occupancy, combined with changes in the transcriptional factor milieu as maturation proceeds, may contribute to transcriptional repression and negative chromatin remodeling.
As human erythroid development proceeds, the proper β-like globin genes are activated or repressed, giving rise to the different hemoglobin chains expressed throughout development. Fetal hemoglobin (γ-globin) is silenced shortly after birth, and the adult hemoglobins (β- and δ-globin) are activated reciprocally. However, the γ-globin genes remain in a “transcription-ready” state, since they can be reactivated following inducing treatments such as hydroxyurea or 5-azacytidine, or by naturally occurring HPFH mutations. It is possible that the loss of GATA-2 occupancy after day E12 at the −566 Aγ-globin GATA site () results in the simultaneous loss of transcriptional co-activators associated with GATA-2, dictating the initial event in the onset of γ-globin silencing (). Thus, the change in GATA occupancy, from GATA-2 during early fetal definitive erythropoiesis to GATA-1 at late fetal definitive erythropoiesis observed at this site may be orchestrated by an alteration in the nearby chromatin, post-translational modification of proteins and/or changes in the transcription co-factors available in the neighborhood ().
The demonstration of co-localization of GATA-1, FOG-1, and Mi2 by ChIP does not prove interaction between those proteins. Since we are analyzing a small region in the more distal promoter region of the A
γ-globin gene, it is possible that these proteins are associated with other complexes in the neighborhood, but still detected by ChIP due to the cross-linking step and size of the fragments after sonication. Hence, we do not exclude the hypothesis that other transcription factors and cofactors are recruited to nearby sites and contribute additively to silencing. Factors such as BCL11A, the orphan nuclear receptors TR2 and TR4, NF-3/COUP-TFII and Ikaros have been associated with γ-globin silencing 
. More recently, Ikaros was shown to interact with GATA-1, since a lack of Ikaros reduced GATA-1 binding at the γ-globin promoter and delayed γ-globin gene silencing 
. This study demonstrated that Ikaros functioned in the silencing of γ-globin by recruiting a repressor complex containing GATA-1, FOG-1, Mi2 and HDAC1.
Overall, the data presented in this study provide clear evidence of the involvement of GATA-1 and Mi2 in silencing γ-globin gene expression. In a recent study, Miccio and Blobel 
used mutant mice expressing an altered FOG-1 that abrogated NuRD binding. The authors demonstrated that the FOG-1/NuRD interaction is dispensable for silencing γ-globin expression, but is required for FOG-1-dependent activation of human adult globin expression 
. These data do not discriminate whether these proteins directly interact to form a mega-complex, with repressive and activator protein partners, or if a sub-population of the proteins interacts to form a distinct repressor complex and another sub-population interacts to form a distinct activator complex. A deficiency of Ikaros reduced GATA-1 binding at the A
γ-globin promoter, enhanced chromosomal proximity between the LCR and the A
γ-globin promoter and delayed γ-globin silencing. An Ikaros-related consensus binding sequence is found at the −566 position of the A
γ-globin gene 
, thus it is provocative to suggest that Mi2 associates with Ikaros and GATA-1 to form a fetal γ-globin repressor complex that also contains FOG-1 (). However, GATA-1-FOG-1 may interact with a different NuRD component, such as MTA1, and perhaps other NuRD subunits, to form an adult β-globin activator complex 
. A significant reduction of adult-type human and murine β-like globin gene expression was observed in the bone marrow of adult β-YAC transgenic mice when the FOG-1/NuRD interaction was disrupted, suggesting that NuRD is required for FOG-1-dependent activation of adult globin gene expression 
. Bowen et al. suggested that the Mi2/NuRD complex is, in fact, a set of distinct complexes with similar biochemical properties 
. The existence of different NuRD complex sub-types could explain the distinct roles and functions of the NuRD complex in globin regulation. One sub-type complex might be associated with activation of the adult β-globin gene and another sub-type, with shared, but also unique subunits, might be associated with repression of γ-globin gene expression. Finally, Gnanapragasam et al. demonstrated that transient knockdown of p66α and Mi2β induced γ-globin expression by 6- and 8-fold, respectively, in CID-dependent β-YAC bone marrow cells 
, which supports our data showing that Mi2 is required for γ-globin silencing.
Finally, our studies also show that maintenance of γ-globin expression observed with the −566 A
γ-globin HPFH point mutation resulted from the disruption of GATA-1-FOG-1-Mi2-mediated repression (). This finding was corroborated by the increased expression of γ-globin in the Mi2β conditional knockout lines (). Although the HPFH phenotype produced by the −566 A
γ-globin HPFH point mutation was weak, it was still at a level therapeutic for the treatment of hemoglobinopathies 
. Heterocellular HPFH represents approximately 10% of the F cell trait population, with HbF levels between 0.8 and 5% 
. The modest levels of γ-globin produced by the −566 A
γ-globin HPFH might be characteristic of a heterocellular HPFH, as demonstrated by cytospin preparations of RBCs (). In contrast, the Mi2β conditional knockout resulted in a pancellular HPFH (). The Mi2β knockout has a broader effect within RBCs than the cis
-linked −566 A
γ-globin HPFH mutation; the loss of Mi2β may generally affect a number of γ-globin repressive mechanisms, leading to a pancellular F cell distribution, whereas the −566 mutation variably affects binding of a single γ-globin repressor complex, producing a heterocellular distribution. Data from HFPH patients bearing a mutation at the −567 G
γ-globin GATA motif also suggested variance in the levels of HbF caused by the point mutation. Chen et al. 
demonstrated that the father and his 9-year-old son had moderately elevated Hb F at 10.2% and 5.9%, respectively 
. The variance in the levels of γ-globin observed between different −566 A
γ-globin HPFH β-YAC transgenic animals from individual lines suggests position effect variegation (PEV) is operative. Bottardi et al. 
demonstrated that interaction between the LCR and the A
γ-globin gene is reduced by binding of Ikaros to the A
γ-globin promoter at the time of the γ- to β-globin switch. Thus, the chromatin organization of the γ-globin promoter might be essential to maintain the long-range interaction with the LCR. The presence of the −566 point mutation may prevent the promoter from fully interacting with the LCR, blocking full engagement with the LCR necessary for complete transcriptional activation, resulting in PEV.
In conclusion, our study is the first to demonstrate the temporal assembly of a GATA-1 repressor complex in vivo. We also demonstrated that the temporal repression mechanism is disrupted by a Mi2β mutation or a HPFH mutation, alleviating the stage-specific silencing of the Aγ-globin gene by the GATA-1-FOG-1-Mi2 repressor complex. This mechanism potentially provides a new target for treatment of sickle cell disease and other hemoglobinopathies.